RU2035187C1 - Method for tracing of bacterial antigen - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: method involves passive hemagglutination by mixing examined antigen with erythrocytar diagnosticum. Said mixing is carried out in electrolyte which is used as reaction medium. Mentioned above mixture is placed into microchamber and is insulated of electrolyte by agarose. Microchamber is displaced in electrolyte, reaction is carried out at field intensity 700-1000 V/m, current density being 0,1-0,2 А/mm². Results are analyzed with the help of microscope. EFFECT: improves efficiency of the method. 1 dwg

Description

 The invention relates to medical microbiology, in particular to methods for the rapid indication of bacterial antigens. It can be used in the event of a focus of bacterial infection, as well as in cases where it is required to obtain results as soon as possible.

Known methods for indicating bacterial antigens using the luminescent-serological method, which includes the preparation of smears from samples of objects, their subsequent drying, fixing in alcohol, processing for 20 minutes with specific luminescent serums, followed by sequential washing with phosphate-buffered saline solutions for 10 minutes, drying slides and viewing smears under a luminescent microscope [1]
This method is time-consuming when viewing a large number of drugs, highly specific luminescent serums are used in the research process, considerable time is required from the start of the study to obtaining the results.

Closest to the proposed invention is a method of staging a passive hemagglutination reaction in the indication of bacterial antigens, which includes introducing an erythrocyte diagnosticum into a cultured test material and monitoring the formation of complexes of bacterial antigens with erythrocyte diagnosticums on the bottoms of containers in the form of a typical picture of the end of the reaction [2]
The disadvantage of this method is the duration of the sedimentation formation of a typical pattern of erythrocyte sedimentation on the surface of the bottoms of the wells, since the size and shape of the outline of the erythrocytes deposited on the bottom of the wells take into account the reaction results, and the sedimentation sedimentation time of the red blood cells is 2-4 hours and does not meet the requirements of the accelerated diagnosis of microorganisms .

 The essence of the invention lies in the fact that the method of indicating bacterial antigens involves setting up a passive hemagglutination reaction, mixing the test antigen with an erythrocyte diagnosticum in the reaction medium, and taking into account the results of the reaction.

An electrolyte is used as a reaction medium, a mixture of erythrocyte diagnosticum and the studied antigen is placed in a microchamber, isolated from the electrolyte by agarose, the microchamber is placed in the electrolyte with insulating conductors perpendicular to the electric field, the reaction is carried out at an electric field of 700-1000 V / m and current density 0, 1-0.2 A / mm ² , and the results are kept under a small magnification of the microscope.

Distinctive features of the invention are that an electrolyte is used as a reaction medium, a mixture of erythrocyte diagnosticum and the test antigen is placed in a microchamber, isolated from agarose from the electrolyte, the microchamber is placed in the electrolyte with insulating conductors perpendicular to the electric field, the reaction is carried out at an electric field of 700-1000 V / m and current density of 0.1-0.2 A / mm ^2, and the results are kept under a small magnification of the microscope.

 The method is as follows.

Tris-acetate buffer solution pH 8.0 in the required volume of 0.2 or 0.4 ml is added to the wells of the polystyrene plate or to the tubes, the test material is titrated with a multiplicity of 2, then the required material is added to each well or tube according to the method settings (by micro- or macro-methods) the number of erythrocyte diagnosticum, which, depending on the nature of the test material, can be immunoglobulin or antigenic. Microcameras, i.e. the capillary tubes used to carry out the method are filled with contents from each well or tube after being lightly shaken. As microchambers, one can use capillaries from the set “Diagnostic Serum for C-Reactive Protein”, manufactured by the Antigen bacterial production plant in Elektrogorsk. Microcameras filled with the studied material are placed on the table in a small horizontal electrophoretic chamber, the dimensions of which allow it to be placed on the microscope stage. The end openings of the microchambers are oriented towards the electrodes. Facing towards the electrodes microcameras end openings closed with a 1% agarose solution at a temperature of 45-50 ^{° C,} and the electrophoretic chamber is filled with Tris-acetate buffer, pH 8.0 while tension is 700-1000 V / m and the current density 0.1-0.2 A / mm ² . The capillaries are coated on top with a buffer about 1 mm thick. With positive results of the reaction in microchambers under a small increase after 2-3 minutes from the beginning of the supply of voltage and current, within the above parameters, the formation of a typical pattern of erythrocyte hemagglutination in the form of large "flakes" consisting of 12-15 red blood bodies with full background illumination is noted in comparison with the uniform distribution of red blood cells in the volume of control microchambers, i.e. with control diagnostics.

 The drawing shows graphs of the dependence of the rate of formation of hemagglutinating complexes on the strength and current density.

 Graph 1 expresses the dependence of the rate of formation of a typical hemagglutination pattern in the volume of the experimental microchamber from the tension expressed in V / m.

Graph 2 expresses the dependence of the rate of formation of a typical hemagglutination pattern in the volume of the experimental microchambers on the current density, expressed in A / mm ² .

Conclusions: when the electric field strength is in the range of 700-1000 V / m, the current density is 0.1-0.2 A / mm ² , the optimal dependence of the formation of hemagglutinating complexes on time is noted. The points of intersection of the lines of graphs 1 and 2 with each other and their lengths within certain time intervals (2-3 minutes) outline a geometric figure (shaded segment) of a certain area, including a combination of the most optimal parameters of tension, current density and time used in the method. The extreme right intersection point of graphs 1 and 2 limits the maximum values of the parameters of intensity and current density, the excess of which leads to the destruction of hemagglutinating complexes.

 Thus, in the electric field under the influence of electromotive forces arising in the electric field due to the presence of surface charges in erythrocytes of diagnosticums and in antigens, the contacts of species-specific antibodies and antigens are accelerated, the time taken to account for the results of the reaction, and therefore the diagnosis, is reduced. In addition, it is possible that in the electric field there is an increase in the interaction of specifically species-specific antibodies and antigens.

 The method for indicating bacterial antigens is illustrated by the following examples using surface swabs prepared and infected with cells of the vaccine strain Y. pestis EV as diagnostic objects and using a diagnosticum of 2.5% plague immunoglobulin erythrocytes.

PRI me R 1. To the test material flushing from the surface, infected with cells of the vaccine strain Y. pestis EV at a concentration of 0.20 ^. 10 ⁷ μm / ml, 1% formalin was added for disinfection. Flushing from the surface in a volume of 0.4 ml was introduced into the initial well of a polystyrene plate containing 0.4 ml of diluent liquid, which was used as Tris-acetate buffer solution, pH 8.0, and titrated in 6 wells. One drop of 2.5% plague immunoglobulin erythrocytes was added to each well with the cultivated test material, which were evenly suspended in liquid by light shaking of the plate. In a bottle with a dry red blood cell diagnosticum, a Tris-acetate buffer solution in the indicated volume was also previously added. Glass capillary tubes 10-15 mm long were alternately filled with the contents of each well and laid on a horizontal table of the electrophoretic chamber with the orientation of the end openings of the microchambers, i.e. capillaries in the direction of the electrodes. Closed end openings of the capillaries 1% solution of agarose in tris-acetate buffer, pH 8.0 at a temperature of 47 ^C. The horizontal electrophoretic chamber was filled with tris-acetate buffer pH 8.0 was set to the microscope stage, found in the view of the studied objects (capillaries) erythrocytes at low magnification. In this case, the buffer solution covered the capillaries with a thickness of about 1 mm. The voltage at the electrodes of the electrophoretic chamber was 600 V / m at a current density of 0.1 A / mm ² . The reaction was taken into account after 3 minutes and at the same time in the field of view of each of the six capillaries there was no formation of “flakes” in the form of dense clusters of red blood cells in the amount of 12-15 with a background brightening. A further increase in the observation time was accompanied by changes in the erythrocyte hemagglutination pattern, which clearly appeared after 4 min of observation in the field of view of each capillary, while in capillaries prepared from wells with a microbe concentration of 62 thousand μ / ml and 31 thousand μ / ml, areas in the fields of vision of which there were clearly defined hemagglutinates.

In the field of view mounted on a capillary containing control red blood cells with the highest concentration of antigens 0.20 ^. 10 ⁷ μ / ml in Tris-acetate buffer solution, based on one drop of 2.5% concentration in 0.4 ml, for 3 minutes or longer up to 30 minutes of observation with an electric field with the indicated intensity and current changes in the picture, the distribution of red blood cells was not noted.

The reliability of taking into account the positive result of hemagglutination in an electric field in capillary tubes was checked when examining flushing material from a surface prepared under the same conditions, but not infected with the cells of the vaccine strain Y.pestis EV. Flushing from the surface in a volume of 0.4 ml was introduced into the initial well of a polystyrene plate containing 0.4 ml of diluent liquid, which was used Tris-acetate buffer solution, pH 8.0, and titrated in six wells. In a bottle with a dry erythrocyte diagnosticum, Tris-acetate buffer was previously added in the indicated volume. One drop of 2.5% plague immunoglobulin erythrocytes was added to each well with the cultivated test material, which were evenly suspended in liquid by light shaking of the plate. Glass capillary tubes 10-15 mm long were alternately filled with the contents of each well and laid on a horizontal table of the electrophoretic chamber with the orientation of the end openings of the microchambers, i.e. capillaries in the direction of the electrodes. The end openings closed capillaries 1.0% solution of agarose in tris-acetate buffer solution, pH 8.0 at a temperature of 47 ^C. The electrophoretic chamber was filled with tris-acetate buffer, pH 8.0, set it on the microscope stage, erythrocytes were found in the field of view of the studied objects (capillaries) at low magnification. In this case, the buffer solution covered the capillaries with a layer about 1 mm thick, the voltage at the electrodes was 600 V / m, and the current density was 0.1 A / mm ² . The reaction was taken into account after 2.5 minutes, while in the field of view of each of the six capillaries no formation of "flakes" in the form of dense clusters of red blood cells in the amount of 12-15 with background brightening was noted. Red blood cells were distributed evenly throughout the field of view. A further increase in the observation time was not accompanied by changes, including in the picture of the location of the control red blood cells (i.e., in the reaction of the control diagnosticum with the test material).

To control the proposed method for the indication of bacterial antigens with the above parameters of an electric field of intensity 600 V / m and a current density of 0.1 A / mm ² , surface washings infected with the cells of the vaccine strain Y. pestis EV were examined using RPGA. As a diagnosticum, 2.5% plague immunoglobulin erythrocytes were used. Rinsing off the surface was pre-disinfected with 1.0% formalin. Then, the test material in a volume of 0.4 ml was introduced into the initial well of the polystyrene plate and titrated in six wells in physiological saline. The concentration of microbial cells in the flush was 0.20 ^. 10 ⁷ μm / ml, respectively, in the last sixth well there were 31 thousand microns / ml. The control RPGA was taken into account after 2 hours. In the first three holes, positive results were noted with a score of (++++). In the fourth well, erythrocytes uniformly covered more than 2/3 of the surface area of the bottom of the well, which can be estimated with a sign (++++), and where the cell concentration was 125 thousand μm / ml, which corresponds to the sensitivity specified by the manufacturer. The reliability of taking into account the positive result of RPHA was checked when examining the material of flushing from the surface, prepared under the same conditions, but not infected with the cells of the vaccine strain Y.pestis EV. The material to be studied in a volume of 0.4 ml was added to the initial well of the polystyrene plate, titrated in six wells in physiological saline. Accounting was performed after 2 hours. Moreover, the formation of buttons was noted in all holes. In the control wells of RPHA, i.e. of those where control red blood cells were added, the formation of “buttons” was also noted, i.e. red blood cells rolled down to the center.

Conclusion: this example demonstrates a reduction in the timing of the hemagglutination reaction in an electric field to 4 min at a voltage of 600 V / m and a current density of 0.1 A / mm ² with a simultaneous increase in sensitivity to 31 thousand microns / ml, however, the reaction time is quite long . When setting up the reaction in an electric field with the above parameters and examining the flush that was not infected with Y. pestis EV cells, no picture of the change in the location of red blood cells in the field of view was noted. Negative results were obtained in RPGA, which was set to control the accuracy of accounting with material uninfected with Y. pestis EV cells.

PRI me R 2. To the test material flushing from the surface, infected with cells of the vaccine strain Y. pestis EV at a concentration of 0.20 ^. 10 ⁷ μ / ml, 1.0% formalin was added for disinfection. Flushing from the surface in a volume of 0.4 ml was introduced into the initial well of a polystyrene plate containing 0.4 ml of a diluting liquid, which was used as a Tris-acetate buffer solution pH 8.0, and titrated in six wells. In a bottle with a dry erythrocyte diagnosticum, Tris-acetate buffer was previously added in the indicated volume. One drop of 2.5% plague immunoglobulin erythrocytes was added to each well with the cultivated test material, which were evenly suspended in liquid by light shaking of the plate. Glass capillary tubes with a length of 10-15 mm were alternately filled with the contents of each well and laid on a horizontal table of the electrophoretic chamber with the orientation of the end openings of the capillaries in the direction of the electrodes. End openings of microcameras, i.e. capillaries sealed 1.0% solution of agarose in tris-acetate buffer, pH 8.0 at a temperature of 47 ^C. The electrophoretic chamber was filled with tris-acetate buffer, pH 8.0, was mounted on the microscope stage, found in the field of view of the studied objects (capillaries) erythrocytes at low magnification. In this case, the buffer solution covered the capillaries with a layer about 1 mm thick. At the electrodes of the electrophoretic chamber, the voltage was 700 V / m at a current density of 0.1 A / mm ² . The reaction was taken into account after 3 minutes, while in the field of view of each of the six capillaries the formation of "flakes" in the form of dense clusters of red blood cells in the amount of 12-15 with a background brightening was noted. Moreover, in the capillaries filled from the wells with a concentration of microbial cells of 62 thousand microns / ml and 31 thousand microns / ml, the formation of distinct hemagglutinates was noted not along the entire length of the capillaries, but only in separate sections, which is probably due to the limited amount of antigen. A further increase in the observation time was not accompanied by changes in the picture of hemagglutination of red blood cells. In the field of view mounted on a capillary containing control erythrocytes in Tris-acetate buffer, based on one drop of 2.5% concentration, made in 0.4 ml, for 3 min and longer observation at an electric field of 700 V / m and a current density of 0.1 A / mm ^{2 there were no} visible changes in the red blood cell distribution pattern, i.e. the latter remained uniformly distributed throughout the entire volume of the capillary.

The reliability of taking into account the positive result of hemagglutination in an electric field in capillary tubes was checked when examining flush material from a surface prepared under the same conditions, but not infected with the cells of the vaccine strain Y.pestis EV. Flushing from the surface in a volume of 0.4 ml was introduced into the initial well of a polystyrene plate containing 0.4 ml of diluent liquid, which was used as a Tris-acetate buffer solution pH 8.0, and titrated in six wells. In a bottle with a dry erythrocyte diagnosticum, Tris-acetate buffer was previously added in the indicated volume. One drop of 2.5% plague immunoglobulin erythrocytes was added to each well with the cultivated material, which were suspended in a liquid by light shaking. Glass tubes 10-15 mm long were alternately filled with the contents of each well and laid on a horizontal table of the electrophoretic chamber with the orientation of the end openings of the microchambers, i.e. capillaries towards the electrodes. The end openings closed capillaries 1.0% solution of agarose in tris-acetate buffer pH 8.0 at a temperature of 47 ^C. The electrophoretic chamber was filled with tris-acetate buffer, pH 8.0, set it on the microscope stage, found in the field of view of the studied objects (capillaries) erythrocytes at low magnification. In this case, the buffer solution covered the capillaries with a layer about 1 mm thick, the voltage at the electrodes was 700 V / m, and the current density was 0.1 A / mm ² .

 The reaction was taken into account after 2.5 min, while in the field of view of each of the six capillaries no formation of "flakes" in the form of dense clusters of red blood cells in the amount of 12-15 with background brightening was noted. Red blood cells were distributed evenly throughout the field of view. A further increase in the observation time was not accompanied by changes, including in the picture of the location of control erythrocytes (i.e., in the reaction of control diagnostics with the test material).

To control the proposed method for indicating bacterial antigens using the above parameters of the electric field, an RPHA was performed, in which the surface wash was prepared under the same conditions and infected with the cells of the vaccine strain Y.pestis EV at a concentration of 0.20. ^. 10 ⁷ μm / ml. The material to be studied was pre-disinfected with 1.0% formalin, then in a volume of 0.4 ml, the latter was introduced into the initial well of the polystyrene plate and titrated in six wells in physiological saline. The concentration of microbial cells in the last well was 31 thousand mk / ml. The control RPGA was taken into account after 2 hours. In the first three holes, a positive result was noted with a score of (++++). In the fourth well, erythrocytes uniformly covered 2/3 of the surface area of the bottom of the well, which can be estimated with the sign (++++), and where the concentration of microbial cells was 125 thousand mk / ml, as indicated by the manufacturer, the sensitivity of the diagnosticum.

 The reliability of taking into account the positive result of the RPHA was checked when examining the flush material from the surface, prepared under the same conditions, but not infected with the cells of the vaccine strain Y.pestis EV. The material to be studied in a volume of 0.4 ml was added to the initial well of the polystyrene plate and titrated in six wells in physiological saline. The reaction was taken into account after 2 hours. At the same time, the formation of buttons was observed in all holes. In the control wells of the RPHA (that is, where the control red blood cells were added), the formation of “buttons” was also noted, i.e. red blood cells rolled down to the center.

Conclusion: this example demonstrates a reduction in the timing of the hemagglutination reaction in an electric field to 3 min at a voltage of 700 V / m and a current density of 0.1 A / mm ² with a simultaneous increase in sensitivity to 31 thousand microns / ml, the reaction time in this case quite long. When staging a reaction in an electric field with the above parameters and examining a washout that was not infected with Y. pestis EV cells, no pattern of a change in the uniform distribution of red blood cells in the field of view was detected. Negative results were obtained in RPGA, which was set to control the accuracy of accounting with material uninfected with cells of the vaccine strain Y.pestis EV.

PRI me R 3. To the test material flushing from the surface, infected with cells of the vaccine strain Y. pestis EV at a concentration of 0.20 ^. 10 ⁷ μ / ml, 1.0% formalin was added for disinfection. Flushing from the surface in a volume of 0.4 ml was introduced into the initial well of a polystyrene plate containing 0.4 ml of diluent liquid, which was used as Tris-acetate buffer solution, pH 8.0, and titrated in six wells. In a bottle with a dry erythrocyte diagnosticum, Tris-acetate buffer was previously added in the indicated volume. One drop of 2.5% plague immunoglobulin erythrocytes was added to each well with the cultivated test material, which were evenly suspended in liquid by light shaking of the plate. Glass capillary tubes 10-15 mm long were alternately filled with the contents of each well and laid on a horizontal table of the electrophoretic chamber with the orientation of the end openings of the microchambers, i.e. capillaries in the direction of the electrodes. The capillary end openings were closed with a 1.0% agarose solution in Tris-acetate buffer, pH 8.0 at 47 ^о С. The electrophoretic chamber was filled with Tris-acetate buffer solution, pH 8.0, it was mounted on a microscope stage, and it was found in the field of view of the studied objects (capillaries) erythrocytes at low magnification. In this case, the buffer solution covered the capillaries with a layer about 1 mm thick, the voltage at the electrodes of the electrophoretic chamber was 850 V / m at a current density of 0.15 A / mm ² . The reaction was taken into account after 2.5 min, while in the field of view of each of the six capillaries the formation of "flakes" in the form of dense clusters of red blood cells in the amount of 12-15 with a background brightening was noted. At the same time, in the capillaries filled from wells with a concentration of microbial cells of 62 thousand microns / ml and 31 thousand microns / ml, the formation of pronounced hemagglutinates was noted not along the entire length of the capillaries, but only in separate areas, which is associated with a limited number of antigens. A further increase in the observation time was not accompanied by changes in the picture of hemagglutination of red blood cells. In the field of view installed on a capillary containing control red blood cells in a Tris-acetate buffer solution based on one drop of their 2.5% concentration, made in 0.4 ml, for 3 min or longer observation no changes were noted in the picture of the uniform distribution of red blood cells over the volume of the capillary at a voltage of 850 V / m and current density A / mm ² .

The reliability of taking into account the positive result of hemmagglutination in an electric field in capillary tubes was checked when examining flush material from a surface prepared under the same conditions, but not infected with the cells of the vaccine strain Y.pestis EV. Flushing from the surface in a volume of 0.4 ml was introduced into the initial well of a polystyrene plate containing 0.4 ml of diluent liquid, which was used as Tris-acetate buffer solution, pH 8.0, and titrated in six wells. In a bottle with a dry erythrocyte diagnosticum, Tris-acetate buffer was previously added in the indicated volume. One drop of 2.5% plague immunoglobulin erythrocytes was added to each well with the cultivated test material, which were evenly suspended in liquid by light shaking of the plate. Glass capillary tubes 10-15 mm long were alternately filled with the contents of each well and laid on a horizontal table of the electrophoretic chamber with the orientation of the end openings of the microchambers, i.e. capillaries in the direction of the electrodes. The end openings closed capillaries 1.0% solution of agarose in tris-acetate buffer, pH 8.0, at a temperature of 47 ^C. The electrophoretic chamber filled with Tris-acetate buffer, pH 8.0, set it on the microscope stage , found in the field of view of the studied objects (capillaries) red blood cells at low magnification. In this case, the buffer solution covered the capillaries with a layer about 1 mm thick, the voltage at the electrodes was 850 V / m, and the current density was 0.15 A / mm ² .

 The reaction was taken into account after 2.5 min, while in the field of view of each of the six capillaries no formation of "flakes" in the form of dense clusters of red blood cells in the amount of 12-15 with background brightening was noted. Red blood cells were distributed evenly throughout the field of view. A further increase in the observation time was not accompanied by changes, including in the picture of the location of control red blood cells, i.e. in the reaction of control diagnosticums with the test material.

To control the proposed method for indicating bacterial antigens using the above parameters of the electric field, an RPHA was performed, in which the surface wash was prepared under the same conditions and infected with the cells of the vaccine strain Y.pestis EV at a concentration of 0.20. ^. 10 ⁷ μm / ml. The material to be studied was pre-disinfected with 1.0% formalin, then in a volume of 0.4 ml it was introduced into the initial well of the polystyrene plate and titrated in six wells in physiological saline. The concentration of microbial cells in the last well was 31 thousand mk / ml. The control RPGA was taken into account after 2 hours. In the first three holes, a positive result was noted with a score of (++++). In the fourth well, erythrocytes uniformly covered 2/3 of the surface area of the bottom of the well, which can be estimated with the sign (++++), and where the concentration of microbial cells was 125 thousand mk / ml, which corresponds to the sensitivity of the diagnosticum indicated by the manufacturer.

 The reliability of taking into account the positive result of the RPHA was checked when examining the flush material from the surface, prepared under the same conditions, but not infected with the cells of the vaccine strain Y.pestis EV. The material to be studied in a volume of 0.4 ml was added to the initial well of the polystyrene plate and titrated in six wells in physiological saline. The reaction was taken into account after 2 hours. At the same time, the formation of buttons was observed in all holes. In the control wells of RPHA, i.e. those where control red blood cells were introduced, the formation of buttons was also noted, red blood cells rolled down to the center.

Conclusion: this example demonstrates a reduction in the timing of the hemagglutination reaction in an electric field to 2.5 min at a voltage of 850 V / m and a current density of 0.15 A / mm ² with a simultaneous increase in sensitivity to 31 thousand microns / ml. The reaction time in this case is short. When staging a reaction in an electric field with the above parameters and examining a washout that was not infected with Y. pestis EV cells, no pattern of a change in the uniform distribution of red blood cells in the field of view was detected. Negative results were obtained in RPGA, which was set to control the accuracy of accounting with material uninfected with Y. pestis EV cells.

PRI me R 4. To the test material flushing from the surface, infected with cells of the vaccine strain Y. pestis EV at a concentration of 0.20 ^. 10 ⁷ μ / ml, 1.0% formalin was added for disinfection. Flushing from the surface in a volume of 0.4 ml was introduced into the initial well of a polystyrene plate containing 0.4 ml of diluent liquid, which was used as Tris-acetate buffer solution, pH 8.0, and titrated in six wells. In a bottle with a dry erythrocyte diagnosticum, Tris-acetate buffer was previously added in the indicated volume. One drop of 2.5% plague immunoglobulin erythrocytes was added to each well with the cultivated test material, which were evenly suspended in liquid by light shaking of the plate. Glass capillary tubes 10-15 mm long were alternately filled with the contents of each well and laid on a horizontal table of the electrophoretic chamber with the orientation of the end openings of the microchambers, i.e. capillaries in the direction of the electrodes. The capillary end openings were closed with a 1.0% agarose solution in Tris-acetate buffer, pH 8.0 at 47 ^о С. The electrophoretic chamber was filled with Tris-acetate buffer solution, pH 8.0, it was mounted on a microscope stage, and it was found in the field of view of the studied objects (capillaries) erythrocytes at low magnification. In this case, the buffer solution covered the capillaries with a layer about 1 mm thick, the voltage at the electrodes of the electrophoretic chamber was 1000 V / m at a current density of 0.2 A / mm ² . The reaction was taken into account after 2.0 min, while in the field of view of each of the six capillaries the formation of "flakes" in the form of dense clusters of red blood cells in the amount of 12-15 with a background brightening was noted. At the same time, in the capillaries filled from wells with a concentration of microbial cells of 62 thousand microns / ml and 31 thousand microns / ml, the formation of pronounced hemagglutinates was noted not along the entire length of the capillaries, but only in separate areas, which is associated with a limited number of antigens. A further increase in the observation time was not accompanied by changes in the pattern of hemagglutinated erythrocytes. In the field of view mounted on a capillary containing control red blood cells with the highest concentration of antigens in these experiments, 0.20 ^. 10 ⁷ μ / ml in Tris-acetate buffer solution based on one drop of 2.5% control erythrocytes in 0.4 ml, for 2 min or longer, with an electric field of 1000 V / m and current density 0 , 2 A / mm ^{2 there were no} visible changes in the pattern of uniform distribution of red blood cells, i.e. the latter remained uniformly distributed throughout the entire volume of the capillary.

The reliability of taking into account the positive result of hemagglutination in an electric field in capillary tubes was checked when examining flushing material from a surface prepared under the same conditions, but not infected with the cells of the vaccine strain Y.pestis EV. Flushing from the surface in a volume of 0.4 ml was introduced into the initial well of a polystyrene plate containing 0.4 ml of diluent liquid, which was used Tris-acetate buffer solution, pH 8.0, and was titrated in six wells. In a bottle with a dry erythrocyte diagnosticum, Tris-acetate buffer was previously added in the indicated volume. One drop of 2.5% plague immunoglobulin erythrocytes was added to each well with the cultivated material, which were evenly suspended in liquid by light shaking of the plate. Glass tubes 10-15 mm long were alternately filled with the contents of each well and laid on the horizontal surface of the table of the electrophoretic chamber with the orientation of the end openings of the microchambers, i.e. capillaries in the direction of the electrodes. The end openings closed capillaries 1.0% solution of agarose in tris-acetate buffer pH 8.0 at a temperature of 47 ^C. The electrophoretic chamber was filled with tris-acetate buffer at pH 8.0, it was set on the microscope stage, found in the field of view of the studied objects (capillaries) erythrocytes at low magnification. In this case, the buffer solution covered the capillaries with a layer about 1 mm thick, the voltage at the electrodes was 1000 V / m, and the current density was 0.2 A / mm ² . The reaction was taken into account after 2 minutes, while in the field of view of each of the six capillaries there was no formation of "flakes" in the form of dense clusters of red blood cells in the amount of 12-15 with a background brightening. Red blood cells were distributed evenly throughout the field of view. A further increase in the observation time was not accompanied by changes, including in the picture of the location of control red blood cells, i.e., in the reaction of control diagnostics with the test material.

To control the proposed method for the indication of bacterial antigens, performed with the above parameters, the intensity of 1000 V / m and current density of 0.2 A / mm ² , was put RPGA, in which as the test material used flush from the surface, infected with cells of the vaccine strain Y pestis EV at a concentration of 0.20 ^. 10 ⁷ μm / ml. The material to be examined was pre-disinfected by adding 1.0% formalin, then in a volume of 0.4 ml was introduced into the initial well of the polystyrene plate and titrated in six wells in physiological saline. The concentration of microbial cells in the last well was 31 thousand mk / ml. The control RPGA was taken into account after 2 hours. In the first three holes, a positive result was noted with a score of (++++). In the fourth well, erythrocytes uniformly covered more than 2/3 of the surface area of the bottom of the well, which can be estimated with a sign (++++), and where the cell concentration was 125 thousand mk / ml, which corresponds to the sensitivity specified by the manufacturer. In the control wells of RPHA, i.e., in wells with a maximum concentration of antigens in these experiments, 0.20 ^. 10 ⁷ µ / ml and one drop of 2.5% control red blood cells in 0.4 ml of saline solution, the formation of buttons was noted, i.e. red blood cells rolled down to the center.

 The reliability of taking into account the positive result of RPHA was believed in the study of flush material from the surface, prepared under the same conditions, but not infected with the cells of the vaccine strain Y.pestis EV. The material to be studied in a volume of 0.4 ml was added to the initial well of the polystyrene plate, titrated in six wells in physiological saline. The RPGA was recorded after 2 hours. Moreover, the formation of “buttons” was noted in all the wells, i.e. red blood cells rolled down to the center.

Conclusion: this example demonstrates a reduction in the time taken into account for the hemagglutination reaction in an electric field to 2 min at a voltage of 1000 V / m and a current density of 0.2 A / mm ² with a simultaneous increase in sensitivity to 31 thousand mk / ml. The reaction time in this case is as short as possible, i.e., it is possible to consider the maximum attainable. When setting up the reaction in an electric field with the above parameters and examining the washout that was not infected with the cells of the vaccine strain Y. pestis EV, the pattern of changes in the uniform distribution of red blood cells in the field of view was not noted.

PRI me R 5. To the test material flushing from the surface, infected with cells of the vaccine strain Y. pestis EV at a concentration of 0.20 ^. 10 ⁷ μ / ml, 1.0% formalin was added for disinfection. Flushing from the surface in a volume of 0.4 L was introduced into the initial well of a polystyrene plate containing 0.4 ml of diluting liquid, which was used as Tris-acetate buffer solution, pH 8.0, and titrated in six wells of a polystyrene plate. In a bottle with a dry erythrocyte diagnosticum, Tris-acetate buffer was previously added in the indicated volume. One drop of 2.5% plague immunoglobulin erythrocytes was added to each well with the cultivated test material, which were evenly suspended in a liquid by lightly shaking the plate. Glass capillaries 10-15 mm long were alternately filled with contents from each well and placed on the horizontal surface of the stage of the electrophoretic chamber with the orientation of the end openings of the capillaries in the direction of the electrodes. The end openings of the capillaries capped with 1.0% agarose in Tris-acetate buffer at pH 8.0 at a temperature of 47 ^C. The electrophoretic chamber was filled with tris-acetate buffer pH 8.0, set it on the microscope stage, found in the view of the studied objects, i.e. capillaries, erythrocytes, and under low magnification were observed. In this case, the buffer solution covered the capillaries with a layer about 1.0 mm thick. The voltage at the electrodes of the chamber was 1100 V / m at a current density of 0.25 A / mm ² . The results of the reaction were taken into account after 2 min, while in the field of view of each of the six capillaries, a random shift of the entire mass of red blood cells in the direction of one of the poles was noted, i.e. electrodes, while the formation of hemagglutinates was not observed in any of the capillaries. A further increase in the observation time was accompanied by compaction of the displaced mass in the direction of one of the electrodes. It should be noted that during the first 1.5 minutes of observation, the formation of hemagglutinates in the form of "flakes" also does not occur. In the field of view mounted on a capillary containing control red blood cells with the highest concentration of antigens in these experiments, 0.20 ^. 10 ⁷ μ / ml, when observed for 2 minutes using the above parameters of the electric field, a shift in the mass of red blood cells to one of the poles is also noted.

The reliability of taking into account the positive result of hemagglutination in an electric field in capillary tubes was checked when examining flushing material from a surface prepared under the same conditions, but not infected with the cells of the vaccine strain Y.pestis EV. Flushing from the surface with a volume of 0.4 ml was introduced into the initial well of a polystyrene plate containing 0.4 ml of diluent liquid, which was used Tris-acetate buffer solution, pH 8.0, and titrated in six wells. In a bottle with a dry erythrocyte diagnosticum, Tris-acetate buffer was previously added in the indicated volume. One drop of 2.5% plague immunoglobulin erythrocytes was added to each well with the cultivated test material, which were evenly suspended in liquid by light shaking of the plate. Glass capillary tubes 10-15 mm long were alternately filled with the contents of each well and laid on a horizontal table of the electrophoretic chamber with the orientation of the end openings of the microchambers, i.e. capillaries in the direction of the electrodes. The end openings closed capillaries 1.0% solution of agarose in tris-acetate buffer, pH 8.0, at a temperature of 47 ^C. The electrophoretic chamber filled with Tris-acetate buffer pH 8.0, set it on the microscope stage, erythrocytes were found in the field of view of the studied objects (capillaries) at low magnification. In this case, the buffer solution covered the capillaries with a layer about 1 mm thick, the voltage at the electrodes was 1100 V / m, and the current density was 0.25 A / mm ² . The results of the reaction were taken into account after 2 min, while in the field of view of each of the six capillaries, a random shift of the entire mass of red blood cells to one of the poles was noted. In the field of view mounted on a capillary containing control red blood cells with the highest concentration of antigens, these experiments 0.2 ^. 10 ⁷ μ / ml, when observed for 2 minutes using the above parameters of the electric field, a shift in the mass of red blood cells to one of the poles is also noted.

To control the proposed method for the indication of bacterial antigens, an RPHA was performed in which a surface wash infected with the cells of the vaccine strain Y. pestis EV at a concentration of 0.2 was used as the test material ^. 10 ⁷ μm / ml. The material to be studied was pre-disinfected by adding 1.0% formalin, then in a volume of 0.4 ml was introduced into the initial well of the polystyrene plate and titrated in six wells in physiological saline. The concentration of microbial cells in the last well was 31 thousand mk / ml. The RPGA was recorded after 2 hours. In the first three holes, a positive result was noted with a rating of (++++). In the fourth well, erythrocytes uniformly covered more than 2/3 of the surface area of the bottom of the well, which can be estimated with the sign (++++), where the cell concentration was 125 thousand mk / ml, which corresponds to the sensitivity specified by the manufacturer. In the control wells of RPHA, i.e. in wells with a maximum concentration of antigen used in these experiments, 0.2 ^. 10 ⁷ µ / ml and one drop of 2.5% control erythrocytes introduced, the formation of buttons was noted, red blood cells rolled down to the center of the well.

 The reliability of taking into account the positive result of the RPHA was checked when examining the flush material from the surface, prepared under the same conditions, but not infected with the cells of the vaccine strain Y.pestis EV. The material to be studied in a volume of 0.4 ml was added to the initial well of the polystyrene plate, titrated in six wells in physiological saline. The RPGA analysis was carried out after 2 hours. Moreover, the formation of buttons was observed in all the holes. In the control wells of RPHA, i.e. where control erythrocytes were added, the formation of buttons was also noted; red blood cells rolled down to the center of the well.

Conclusion: this example demonstrates the impossibility of further reducing the reaction time in an electric field, since at a voltage of 1100 V / m and a current density of 0.25 A / mm ^{2, a} complete disruption of the red blood cell distribution pattern in capillaries with experimental and control diagnostics was noted, which is in favor the application of limited parameters of the electric field within the range of 700-1000 V / m and current density of 0.1-0.2 A / mm ² to obtain a positive effect. When setting up the reaction in capillaries in an electric field with a voltage of 1100 V / m and a current density of 0.25 A / mm ² and study of flush material from a surface not infected with the cells of the vaccine strain Y. pestis EV, a random shift in the mass of red blood cells to one of the poles was noted. Negative results were obtained in RPGA, which was set to control the accuracy of accounting with material not infected with Y / pestis EV cells.

 It should be noted that the results of the control experiments in all examples, except 5, are completely correlated with those obtained in RPGA.

To confirm the positive effect of the proposed method for indicating bacterial antigens by conducting a reaction in an electrolyte and in an electric field at a voltage of 700-1000 V / m and a current density of 0.1-0.2 A / mm ^2, cholera and brucellosis immunoglobulin were tested in addition to plague immunoglobulin red blood cells erythrocytes on examples of the detection of the corresponding antigens in washouts from the surface. In addition, plague and tularemia antigenic erythrocyte diagnostics were tested on an example of a study of sera from immunized animals (imitating material from patients). At the same time, the accounting time was reduced to 2-3 minutes, and the accuracy (sensitivity) of the proposed method for indicating bacterial antigens was increased up to 4 times in comparison with the well-known RPGA formulation method. Based on the examples, the advantage of the method for indicating bacterial antigens by conducting a reaction in an electrolyte and in an electric field is shown compared with the known method for detecting bacterial antigens using RPGA.

Claims (1)

METHOD FOR INDICATING BACTERIAL ANTIGENES, including setting up the passive hemagglutination reaction by mixing the test antigen with an erythrocyte diagnosticum in the reaction medium and taking into account the reaction results, characterized in that an electrolyte is used as a reaction medium, a mixture of erythrocyte diagnosticum and the studied antigen is placed in a micromar, and the microne is isolated from the microcite; , the microcamera is placed in the electrolyte with insulating conductors perpendicular to the directivity of the electric field, the reaction oestriasis when the electric field intensity 700 1000 V / m and the current density 0.1 0.2 A / mm ^2, and records results lead under low magnification microscope.

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